Research Techniques Made Simple: Zebrafish as a Model System to Study Skin Biology and Pathology
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Transcript of Research Techniques Made Simple: Zebrafish as a Model System to Study Skin Biology and Pathology
Research Techniques Made Simple:Zebrafish as a Model System to
Study Skin Biology and Pathology
Qiaoli Li and Jouni Uitto
Department of Dermatology and Cutaneous Biology, Jefferson Medical College, and Jefferson
Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, PA, USA
Zebrafish and Embryos
• Vertebrate• Has nearly the same
complement of genes as mammals
• Develop externally• Transparent• Easy breeding• Develop quickly
Zebrafish Skin Development
Reprinted with permission from Li et al. (2011) J Invest Dermatol 131:565-71
Genetic Manipulation of Zebrafish Genome
• Forward genetics Ethylnitrosourea (ENU) or random mutagenesis
• Reverse genetics Morpholino knockdown of specific gene expression
ENU MutagenesisModified from (Dahm, 2005) Encyclopedia of Molecular Cell Biology and Molecular Medicine (Meyers RA, ed) Vol. 15: Wiley-VCH, 593-626, with permission.
Modulation of Gene Expression by Morpholino
25 nt oligo
“Knockdown”
Efficiency of Morpholino Knockdown
EGFPSP6 promoter
pCS2/EGFP vector
MO1ATG
5’UTR target gene
col17a1b/EGFP RNA + col17a1b MO1col17a1b/EGFP RNA
Splice morpholino
8.5hpf
CMO2
500bp
300bp
421bp299bp
forwardabcc6b MO2
reverse
EXON 18 EXON 19
ATG morpholino
8.5hpf 8.5hpf
Reprinted with permission from Li et al. (2011) J Invest Dermatol 131:565-71
Application of Zebrafish for Skin Research
• Wound healing and skin repair• Pigmentation• Epidermal adhesion• Skin diseases• Skin immunity• Drug screening
Advantages
• A freshwater vertebrate with a complement of genes very similar to those of mammals
• Rapid development of transparent embryos, allowing visual inspection of internal organs
• Easy breeding, with a large number of embryos and larvae
• Easy manipulation of gene expression with phenotypic consequences
• A facile and cost-efficient system for large-scale screening
Limitations
• Lack of functional stratum corneum and expression of genes necessary for terminal differentiation of epidermis
• The zebrafish genome database is not complete and contains misjoins and misassemblies
• Paucity of antibodies recognizing zebrafish protein sequences